Table_1_Anatomical identification of a corticocortical top-down recipient inhibitory circuitry by enhancer-restricted transsynaptic tracing.DOCX

Despite the importance of postsynaptic inhibitory circuitry targeted by mid/long-range projections (e.g., top-down projections) in cognitive functions, its anatomical properties, such as laminar profile and neuron type, are poorly understood owing to the lack of efficient tracing methods. To this end, we developed a method that combines conventional adeno-associated virus (AAV)-mediated transsynaptic tracing with a distal-less homeobox (Dlx) enhancer-restricted expression system to label postsynaptic inhibitory neurons. We called this method “Dlx enhancer-restricted Interneuron-SpECific transsynaptic Tracing” (DISECT). We applied DISECT to a top-down corticocortical circuit from the secondary motor cortex (M2) to the primary somatosensory cortex (S1) in wild-type mice. First, we injected AAV1-Cre into the M2, which enabled Cre recombinase expression in M2-input recipient S1 neurons. Second, we injected AAV1-hDlx-flex-green fluorescent protein (GFP) into the S1 to transduce GFP into the postsynaptic inhibitory neurons in a Cre-dependent manner. We succeeded in exclusively labeling the recipient inhibitory neurons in the S1. Laminar profile analysis of the neurons labeled via DISECT indicated that the M2-input recipient inhibitory neurons were distributed in the superficial and deep layers of the S1. This laminar distribution was aligned with the laminar density of axons projecting from the M2. We further classified the labeled neuron types using immunohistochemistry and in situ hybridization. This post hoc classification revealed that the dominant top-down M2-input recipient neuron types were somatostatin-expressing neurons in the superficial layers and parvalbumin-expressing neurons in the deep layers. These results demonstrate that DISECT enables the investigation of multiple anatomical properties of the postsynaptic inhibitory circuitry.

尽管中/长程投射（如自上而下投射）所靶向的突触后抑制环路在认知功能中发挥关键作用，但由于缺乏高效的追踪手段，其解剖学特征（如层分布模式与神经元类型）仍鲜为人知。为此，我们开发了一种结合传统腺相关病毒（adeno-associated virus, AAV）介导的跨突触追踪与远端同源框（distal-less homeobox, Dlx）增强子限制性表达系统的方法，用于标记突触后抑制性神经元。我们将该方法命名为“Dlx增强子限制性中间神经元特异性跨突触追踪技术”（DISECT）。我们将DISECT应用于野生型小鼠中从次级运动皮层（secondary motor cortex, M2）到初级躯体感觉皮层（primary somatosensory cortex, S1）的自上而下皮层-皮层环路。首先，我们向M2注射AAV1-Cre，使Cre重组酶在接收M2输入的S1神经元中表达。其次，我们向S1注射AAV1-hDlx-flex-绿色荧光蛋白（green fluorescent protein, GFP），以Cre依赖的方式将GFP转导至突触后抑制性神经元中。我们成功特异性标记了S1中的受体抑制性神经元。通过DISECT标记的神经元的层分布分析显示，接收M2输入的抑制性神经元分布于S1的浅层与深层。该层分布模式与M2投射轴突的层密度分布相一致。我们进一步通过免疫组织化学与原位杂交对标记的神经元类型进行分类。事后分类结果显示，占主导的自上而下M2输入受体神经元类型为浅层的生长抑素阳性神经元与深层的小白蛋白阳性神经元。上述结果表明，DISECT可用于探究突触后抑制环路的多项解剖学特征。